B. Parvin, D. Callahan, W. Johnston, and M. Maestre
Information and Computing Sciences Division and Life Sciences Division, Lawrence Berkeley National Laboratory
Recent advances in sample preparation indicate that it may be possible to microdissect, retrieve, and PCR amplify small (4-10 kbp) subfragments of single DNA molecules such as yeast artificial chromosomes (YACs) [1]. Recently, single YOYO-1 stained DNA molecules were visualized using fluorescence microscopy after having been stretched, oriented and fixed onto a coated microscope coverslip [1]. This method of sample preparation has been referred to as molecular combing. We have assembled and have begun testing a visual servoing fluorescence microcope system suitable for the manual or automated microdissection of single DNA molecules prepared in this manner.
Single lambda DNA molecules were stretched and oriented on untreated microscope coverslips using a modification of the published method [1]. A chamber for rapid (1-2 min) molecular combing was constructed and the receding menicus was generated by withdrawing liquid from the chamber. Lambda DNA molecules were oriented in the direction of the liquid flow, along the short axis of a 24x30mm coverslip. An open microdissection chamber was designed for an inverted Zeiss Axiovert 135 fluorescence microscope and coverslips containing stretched and oriented lambda DNA molecules were mounted in the chamber. Microcapillaries for retrieval of microdissected (scraped) DNA fragments had a diameter of approximately 2 µ and were shaped and pulled using a program developed on a micropipette puller (Model P-87, Sutter Instr. Co., Novato, CA). Capillaries were mounted on a micromanipulator (Model MP-185, Sutter Instr. Co.) and were positioned manually using a key-pad or automatically using robotic vision techniques. The Sutter micromanipulator is capable of ultrafine motion (0.025µ) in the x, y, and z directions and it was possible to scrape 5 separate, specific regions (approximately 4kbp per scrape) along a lambda DNA molecule (48.5 kbp) that had been stretched to a length of approximately 20µ. In these studies it was possible to see scraped DNA accumulating in the tip of the microcapillary indicating that retrieval of the microdissected DNA is possible. The principle of visual servoing has been demonstrated by robotically moving the microcapillary tip to a specific site along a single molecule. In our implementation, we have detected the DNA molecules with the tubular model, and acquired and tracked the capillary with the deformable contours [2]. The robotic-vision system is self-calibrating and requires minimal user interaction. In conclusion, we have demonstrated precise microdissection of single stretched DNA molecules and accumulation of scraped DNA in the tip of a microcapillary. Future efforts will be directed toward determining the number of scraped fragments required for successful PCR amplification of the accumulated DNA in the microcapillary tip.
Acknowledgement This work is supported by the Director, Office of Energy Research, Office of Computation and Technology Research, Mathematical, Information, and Computational Sciences Division, of the U. S. Department of Energy under contract No. DE-AC03-76SF00098 with the University of California. The LBNL publication number is 38164.
References
[1] A. Bensimon, A. Simon, A. Chiffaudel, V. Croquette, F. Heslot, and D. Bensimon, "Alignment and sensitive detection of DNA by a moving interface", Science 265, 2096-2098 (1994).
[2] B. Parvin, C. Peng, W. Johnston, and M.F. Maestre, "Tracking of Tubular Molecules for Scientific Applications," IEEE Transactions on Pattern Analysis and Machine Intelligence, 17, 800-805 (1995).